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SENIOR THESIS

Optimization of co-immunoprecipitation assays to determine the molecular mechanism modulating direct interactions between potassium channel α-subunit proteins hERG and KvLQT1. Estelle Kim, Louise E.O. Darling, Biological Sciences Department, Wellesley College, 22 May 2015.

This material is copyrighted by Estelle Kim and Louise E.O. Darling, 22 May 2015. 2

Table of Contents: Abbreviations/Key Words………………………………………………………………………...3

Abstract…………………………………………..…………………………………………..…....4

Introduction………………….……………………………………………………………….……5 The Kv11.1 Channel…………………………………………………………………….....7 The Kv7.1 Channel………………...…………………………………………………..…10 Motivation………………………………………………………………………………..12 Scientific Approach………………………………………………………………………14

Materials and Methods…………………………………………………………………………...18

Results…………………………………………………………………………………………....21 Representative co-immunoprecipitation...……………………………………………….21 FLAG-tagged hERG……………………………………………………………………...26 Troubleshooting co-immunoprecipitations……………………………………………....29 Anti-GFP co-IP and non-specific IgG control……………………………………..29 Use of GelCode Blue and PonceauS Staining with co-IP analysis………………..35 Crude extracts vs. supernatant after harvesting cells……………………………...38 Treatment with IBMX + pCPT-cAMP vs. no-treatment……..…………………….…….41

Discussion and Future Directions………………………………………………………………..46

References…………………………………………………………………………………….….56

Acknowledgements………………………………………………………………………………60

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Abbreviations/Key Words: cAMP: cyclic adenosine monophosphate cNBHD: cyclic nucleotide binding homology domain HEK: human embryonic kidney hERG: the human ether-a-go-go related gene and its protein product, a voltage-gated potassium channel alpha subunit involved in LQT2 + IKs: slow delayed rectifier K channel current + IKr: rapid delayed rectified K channel current LQTS: Long QT syndrome KvLQT1: a voltage-gated potassium channel alpha subunit involved in LQT1 PKA: protein kinase A Treatment: IBMX (phosphodiesterase inhibitor) + pCPT-cAMP (membrane soluble cAMP analogue)

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Abstract: KvLQT1 and hERG are the voltage-gated K+ channel α-subunits of the cardiac repolarizing currents IKs and IKr, respectively. These currents function to maintain proper action potential durations in cardiomyocytes to ultimately promote a normal heartbeat. Mutations in KCNQ1 or hERG can lead to a loss of proper function in these currents, resulting in arrhythmias. Previous research in transgenic model organisms demonstrated that interactions between pore mutants of KvLQT1 and hERG exist, resulting in mutual downregulation of their respective currents. In addition, direct protein-protein interactions between wild type hERG and KvLQT1, and more specifically between the COOH-termini of the two proteins, have been established. These interactions have been shown to result in downregulation of the repolarizing currents in heterologous cells. We are specifically interested in the molecular mechanisms of hERG- KvLQT1 interactions which are either abrogated through direct binding of cAMP to the putative cyclic nucleotide homology binding domain (cNHBD) in the COOH-terminus of hERG or by downstream, PKA-mediated effects. In order to delineate the molecular mechanism underlying these interactions, classical biochemical assays were performed on human embryonic kidney (HEK) cells co-expressing hERG and KvLQT1. Co-immunoprecipitation protocols were optimized to recapitulate experiments that demonstrate protein-protein interaction not only with FLAG-tagged constructs, but also with fluorescence-tagged constructs. Intracellular cAMP levels were altered through membrane-permeable cAMP analogs and IBMX, and such treatment seems to decrease interaction between hERG and KvLQT1. Although results from this study are qualitative and not yet conclusive, this work potentially furthers our understanding of the physiological regulation of hERG-KvLQT1 interactions and its implications on cardiac arrhythmias in both healthy and diseased states, as well as characterizes novel interactions between two distinct potassium channel families.

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Introduction:

The heart is an electrically driven pump that secures systemic and pulmonary circulation.

Proper contraction and thereby pumping of the heart is controlled by electrical signals recognized as an electrocardiogram (EKG/ECG) at the organismal level and a cardiac action potential at the cellular level. The shape and duration of the cardiac action potential is governed by the subtle interplay between different channels. These channels are responsible for the movement of sodium (Na+), calcium (Ca2+), chloride (Cl-), and potassium (K+) ions across the plasma membrane depending on different voltages, reflected by different phases, to maintain a regular heartbeat. The standard model of a human cardiomyocyte action potential shows 5 phases that are numbered from 0 to 4 (Figure 1). Phase 4 is the resting membrane potential that remains stable caused by the difference in ionic concentrations and conductances across the cell

+ membrane, predominantly controlled by an inwardly rectifying K (IK1) channel. Phase 0 is the rapid depolarization phase due to the opening of the fast sodium channels causing a sudden

+ + increase in membrane conductance to Na and thus a rapid influx of Na ions (INa) into the cell.

Phase 1 of the cardiac action potential occurs with partial inactivation of the fast Na+ channels.

The transient net outward current causing the small downward deflection of the action potential is due to the movement of K+ and Cl- ions. Phase 2, or the "plateau" phase, of the action potential is sustained by a balance between inward movement of Ca2+ ions and outward

+ + movement of K through the slow delayed rectifier K channel (IKs). During phase 3, the "rapid

2+ repolarization" phase of the action potential, the Ca channels close, while IKs remain open as more potassium leak channels open. This ensures a net outward positive current, corresponding to negative change in membrane potential, thus allowing more types of K+ channels to open. 6

+ These are primarily the rapid delayed rectifier K (IKr) channels and IK1. This net outward, positive current (equal to loss of positive charge from the cell) causes the cell to repolarize.

Figure 1. The standard model of the human cardiomyocyte action potential shows the ion channels and currents present at each phase. Phase 4: resting membrane potential. Phase 0: rapid depolarization. Phase 1: small downward deflection. Phase 2: plateau phase. Phase 3: rapid repolarization. (29)

As these ion channels are responsible for a proper action potential, genetic mutations affect the electrical system of the heart in every phase. However, prolongation of the action potential is reflected in ventricular repolarization such that when its rhythm or function is severely disrupted, there are possible effects of syncope, arrhythmia, and sudden cardiac arrest

(10, 27). Additionally, while only one type of sodium channel is responsible for depolarization, there are seven potassium currents regulated by five potassium ion channels involved during the repolarization of the action potential. Thus, dysfunction of specific potassium channels responsible for the currents involved with cardiac repolarization ultimately cause irregular contractions of the heart. More specifically, delayed rectifier K+ currents (IK), composed of the rapidly activating IKr and the slowly activating IKs, are accountable for maintaining the proper repolarization of the cell, associated with terminating the cardiac action potential and thereby affect the heart’s contraction. 7

Kv11.1 and Kv7.1 channels conduct the IKr and IKs currents, respectively (21), which are critical in correctly timing the repolarization of the cell membrane during the cardiac action potential. Both channels are voltage-gated potassium channels present in the cell membrane of cardiac tissues. As voltage-gated channels, they possess a voltage sensor that detects changes in the membrane potential which regulates the passage of potassium ions (21).

The Kv11.1 Channel:

In this study, KCHN2, the official name for the human ether-á-go-go related gene, will be coined to refer to the gene, hERG as the protein, Kv11.1 as the fully assembled channel complex, and IKr as the native current (as taken from 17). KCNH2 is a gene located on human chromosome 7 which was identified to be the basis of chromosome 7-associated long QT syndrome (10). KCNH2 encodes the alpha subunit of hERG which is an 1159 amino acid protein that consists of six transmembrane regions that make up the voltage sensor domain, a pore loop that consists of the pore domain, as well as a cytosolically located amino (NH2-) and carboxyl

(COOH-) termini (10, 26). At the COOH terminus, the cyclic nucleotide binding domain (cNBD) is thought to share homology with the cNBD of canonical voltage-gated K+ channels and hyperpolarizatoin activated channels (38). However, since the role of the cNBD is not completely understood, it will be referred to as the cyclic nucleotide binding homology domain

(cNBHD) from now forward. Structurally, the Kv11.1 channel is comprised of four identical alpha subunits of hERG, which form the channel’s pore as the pore domains from each of the four subunits line the K+ ion conduction pathway (26). Although it has been proposed that there are beta-subunits that seemingly interact with hERG, there is no evidence that they are essential to activate the IKr current. Contenders for subunits that may be essential for human ventricular 8 repolarization are minK (encoded by KCNE1) and MiRP1 (encoded by KCNE2) (2). These two proteins are single transmembrane domain peptides that can co-assemble with hERG in heterologous systems. MinK seemingly increased hERG currents by an unknown mechanism whereas MiRP1 altered hERG current density and gating (2). However, beyond these studies, no consensus has been reached as to the precise extent of these effects.

As a voltage-gated ion channel, the Kv11.1 channel can be present in closed, open, or inactivated states which underlie the physiological role of IKr (26, 37). Transitions between each state are voltage dependent, such that the transition between closed and open states is slower than the transition between open and inactivated states. When repolarization commences, IKr channels recover from inactivation, which in turn passes more current to accelerate repolarization of the

Kv11.1 channel (26, 37). Thus, repolarization at this stage is relatively rapid. At the end of repolarization, IKr channels close slowly until the membrane potential reaches its resting state. 9

Figure 2. A. Representative structure of the alpha subunit of hERG. (16) B. Homotetrameric form of hERG that forms the Kv11.1 channel 3-dimensionally, C. 2-dimensionally. (36).

Furthermore, hERG protein is modified by asparagine (N)-linked glycosylation which is required for proper trafficking to the surface of the cell membrane (14, 23). The protein sequence contains two putative N-linked glycosylation sites in the extracellular region (N598,

N629) (14, 23). However, site-directed mutagenesis has determined that N598 is the main site for N-linked glycosylation (14). Supporting N598 as the only glycosylated site, two bands of hERG have been routinely detected at 135kDa and 155kDa on a western blot (11, 23, 41). The higher molecular mass represents the mature, fully glycosylated form of the hERG protein located in the plasma membrane (23). The smaller molecular mass band is the core-glycosylated, precursor form of hERG located in the endoplasmic reticulum or forward trafficking 10 compartments of the endomembrane system (23). Although the abolition of hERG glycosylation does not prevent complete trafficking to the surface membrane, the molecular mechanisms of N- linked glycosylation have suggested that there is loss of hERG channel stability (14). In addition to the glycosylation of hERG, phosphorylation has been shown play an important role during biogenesis. In particular, hERG contains four putative phosphorylation sites for protein kinase A

(PKA) that is activated by an increase in cyclic AMP (cAMP) levels (9). This PKA activation is reported to be the result of stimulating the beta-adrenergic pathway in the heart in vivo (26). The beta-adrenergic regulators are critical regulators of cardiac function in both normal and pathophysiological states (24). Under normal conditions, beta-adrenergic regulators and their signaling pathways modulate myocardial contraction and relaxation; however, in chronic heart disease, sustained activation of beta-adrenergic signaling pathways perturb normal regulation of the cAMP/PKA activity which ultimately has negative consequences (24). These changes, in consequence, influence IKr that effectively disturb normal cardiac electrical activity.

The Kv7.1 Channel:

Similar to the terminology above, KCNQ1will be coined to refer to the gene, KvLQT1 as the protein, Kv7.1 as the fully assembled channel complex, and IKs as the native current (as taken from Vandenberg et al, 2012). KvLQT1, located on chromosome 11, is a 676 amino acid protein of which the alpha subunit is encoded by KCNQ1 (Figure 3, 26). Structurally similar to hERG,

KvLQT1 is made of six membrane-spanning domains, two intracellular domains, and a pore loop

(26). Four identical subunits conglomerate to form the pore of the Kv7.1 channel through which

+ K ions flow (26). Unlike hERG, KvLQT1 mediates IKs that also contributes to the repolarization of the cell (21). As an IKs current, the Kv7.1 channel progressively opens with an increase of 11 membrane depolarization which gives rise to slowly activating and deactivating K+ currents (28).

Unlike hERG’s potential but unclear interaction with beta-subunits (1, 2), KvLQT1 has shown studies with more definitive evidence. It was reported that injection of minK cDNA gave rise to current similar to that of IKs in Xenopus oocytes, this prompted further investigation which led to results that coexpression of KCNE1 and KCNQ1 provided the proper IKs current. To conduct IKs current, KvLQT1 assembles with its known beta-subunit, minK (KCNE1) (34).Currently, the stoichiometry of the interaction is not definitive as consensus seems to be 2 minK for 4 KvLQT1 alpha-subunits, but there is also evidence for a 1:1 ratio (34). When assembled with minK, the

KvLQT1 channel activates more slowly and at a more positive membrane potential (29).

KvLQT1 in its monomeric form has been regularly detected via immunoblot at about 75kDa (11,

12, 13, 40).

Figure 3. A. Representative structure of the alpha subunit of KvLQT1. (16). B. Homotetrameric form of Kv7.1 complex with MinK channel. (17).

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Motivation:

The Darling Lab is focused on the function, regulation, and biogenesis of potassium ion channel proteins, hERG and KvLQT1, associated with the repolarization currents, IKr and IKs, respectively. As normal cardiac repolarization results from a complex interplay of multiple potassium ion channels, it is conceivable that there would be some functional redundancy across the channels, as the concept of “repolarization reserve” (27). This concept embodies the idea that the inability to conduct one current will not have complete repolarization failure (27). Instead, the remaining potassium currents may still provide sufficient repolarization to compensate for the loss of function (27). Despite the intuitive nature of this proposed “repolarization reserve”, the pore mutants of hERG and KvLQT1 have been reported to mutually downregulate currents in ex vivo experiments from transgenic rabbit models (7, Figure 4). Both transgenes caused downregulation of the complementary K+ currents without altering the stability of the native polypeptides (7). This functional, mutual downregulation prompted investigations whether the interactions between hERG and KvLQT1 may be directly interacting in heterologous stable cell lines (7, 22, 25). Furthermore, heterologous cell lines served to be more simplistic and practical models to ask some fundamental biochemical and biophysical questions about potential hERG-

KvLQT1 interactions (7,22, 25). 13

Figure 4. Pore mutant rabbit models of hERG and KvLQT1 mutually downregulate currents. Experiments were conducted through electrophysiology. LMC is the normal current, LQT2 refers to hERG mutant, and LQT1 refers to KvLQT1 mutant. (7).

Interestingly, deletion of the NH2-terminus of KvLQT1 did not terminate the effects of mutual downregulation; thus, it has been suggested that the interactions between the two channels were mediated at their COOH-termini (11, 22, 25). Although interactions between the two potassium ion channel proteins have been suggested and experimentally demonstrated, the mechanisms by which the interactions occur are not fully understood. It has been suggested that direct protein-protein interactions are mediated by the COOH-termini of each channel subunit through the direct binding of the ubiquitous intracellular signaling molecule cyclic adenosine monophosphate (cAMP) to the cNBHD located in the COOH-terminus of hERG (21). Many studies agree that the second messenger cAMP regulates the hERG potassium channel both 14 directly and indirectly where activating cAMP-dependent protein kinase causes phosphorylation of hERG (8, 9). Ultimately, this leads to a rapid reduction in current density, acceleration of voltage-dependent deactivation, depolarizing shift in voltage-dependent activation, and hERG protein abundance (9). Despite this phenomenon, it has been argued that cNBHD does not have the structural and functional properties to directly bind to the cAMP on hERG using solution

NMR spectroscopy (5, 6, 15, 20). Therefore, we are interested in elucidating the possibilities of how the interactions occur between hERG and KvLQT1.

Scientific Approach:

hERG and KvLQT1 have been successfully detected through immunoblot analysis in previous studies (22, 25) and in our lab, which establishes our western blot methodology before assessing the interactions between the two proteins. Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells coexpressing hERG and KvLQT1 were used as heterologous model systems as they are preferred for the production of proteins in research (3). Although

CHO cells tend to be the standard model system, transient gene expression in HEK cells has been considerably of interest due to its ability to deliver higher expression of protein over the

CHO cells (4). Accordingly, our lab has also seen similar trends and for this reason, we have used HEK cells as our main model system. The plasmid constructs that code for these proteins included either a FLAG tag or GFP tag at the NH2-terminus and COOH-terminus, respectively.

Using these genetically encoded markers, proteins were detected using antibodies or fluorescence microscopy, respectively. To heterologously express these proteins, cells were transiently co-transfected with DNA plasmids that encode hERG and KvLQT1 with the tags described above. 15

Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. More specifically, co- immunoprecipitation (co-IP) is a pull-down assay which uses a specific antibody to target a known protein, possibly through an epitope tag, within a complex of proteins. When pulling down the entire complex of proteins out of solution, unknown members can be identified by further analysis of immunoblot detection. Accordingly, protein-protein interactions can be observed through the binding of two proteins that are thought to be interacting with one another.

In fact, previous studies showed that the interactions between hERG and KvLQT1 were specific through the methods of Surface Plasmon resonance (25). Without the presence of other proteins, only hERG and KvLQT1 were interacting in such assay (25). In addition, it was shown that the proteins were interacting with one another through the use of co-IPs (25). By utilizing Kir2.1 as a pseudonegative control, only hERG and KvLQT1 were shown to be binding to one another (25).

This concept was effectively used prior to immunoblotting to examine the direct and specific protein-protein interactions between hERG and KvLQT1. Through detection of the two potassium ion channel proteins in the pull-down assay, we not only established detectable levels of protein expression, but also showed that they were interacting with one another. Therefore, co-IPs have been and continue to be effective in providing evidence of protein-protein interactions between hERG and KvLQT1.

In particular, we are expecting to see reduction in protein-protein interactions when cells are treated with chemicals IBMX and pCPT-cAMP (22). IBMX is a phosphodiesterase inhibitor that prevents the breakdown of cAMP, and pCPT-cAMP is a membrane permeable cAMP analogue, both reagents work together to elevate intracellular cAMP to hypothetically disrupt the interactions between hERG and KvLQT1 (8, 9, 22). Other chemicals such as forskolin and other 16 cAMP analogues have been used to elevate cAMP levels; however, in this study, we have mainly used IBMX and pCPT-cAMP. Using acceptor photobleach Forster Resonance Energy Transfer

(apFRET) experiments, a quantitative, microscopy method for measuring protein interactions, it has been demonstrated that increasing intracellular cAMP levels reduced the interactions (18, 22).

Although apFRET results seem to address the molecular modulation of the cNBHD of hERG, such experiments are equipped with the restrictions of the limitations that come with fluorescent microscopy, which include specificity to certain regions of the cells and functionality of the proteins. Moreover, although the C-terminal placement of fluorescent labels does not appear to interfere withe the normal function of the proteins (as measured electrophysiologically) or their interactions (as measured via apFRET), co-IPs between fluorescent protein-tagged hERG and

KvLQT1 have not been demonstrated (22). Thus, the traditional biochemical assay will further investigate the role of the hERG cNBHD. Therefore, we predict that detection of protein expression following co-immunoprecipitation will decrease with treatment which will be indicative of the reduction in hERG-KvLQT1 interactions. As we hypothesize that cAMP will bind to the cNBHD, we expect a physical interference preventing the interactions between hERG and KvLQT1. However, the elevation of cAMP levels does not directly reflect the role of the cNBHD as it is not specifically targeting the site of the hERG domain; therefore, mutant CNBD models through site-directed mutagenesis will further deepen our understanding of the interactions at the cNBHD of hERG. By expressing functional and non-functional CNBD mutants (Amanda Papakryikos ’14; Medeea Popescu’17), the same treatment will be induced. In the case that the cNBHD is directly involved in the protein-protein interaction of hERG and

KvLQT1, it is expected that there will be no difference in protein interaction levels when hERG cNBHD mutants are expressed. 17

In addition to understanding the role of the cNBHD, we are interested in prolonging exposure times of elevated cAMP levels. This is especially essential to understand the chronic effects of stress of diseased states of the heart. Other labs interested in cAMP effects on channel function have shown differences in surface expression with acute (<1hr) versus long-term (hours to days) treatment (8). When treated with IBMX and pCPT-cAMP over a long period of time, it is expected that hERG expression increases effectively rescuing mutual, functional downregulation (8). It has been shown that in Xenopus oocytes, chronic stimulation of cAMP increased current density, likely through increased surface expression of hERG proteins (19); yet, the mechanism was not fully understood. In such case, we will be able to indicate such changes by detecting higher levels of expression of the mature, glycosylated form as well the immature, trafficking form of hERG through western blot analyses. These acute and prolonged regulation of cAMP levels reflect the acute and chronic beta-adrenergic stimulation to which may prove to show a mechanistic link between such signaling and sudden death in acquired heart disease as well as genetic cases. Understanding these mechanisms will enable the examination of the physiological regulation of the protein-protein interactions between KvLQT1 and hERG and its implications on cardiac rhythms in both healthy and diseased states.

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Methods:

Transfection of HEK-293 Cells:

Human embryonic kidney 293 (HEK-293, ATCC CRL-1573) cells were cultured at 37oC with 5% CO2 in Eagle’s Minimum Essential Medium (ATCC) with 10% FBS (Thermo Scientific

Pierce) and 1% penicillin-streptomycin (Thermo Scientific Pierce), which comprises complete cell medium. HEK-293 cells were plated on 10-cm dishes (CellTreat) at 1.5-2 million cells/dish.

Transient transfections into HEK-293 cells were performed following the manufacturer’s instructions using Xtreme-GENE9 (Roche Applied Science): DNA ratio of 2uL:1ug. Co- transfections for co-immunoprecipitation used equal amounts (1ug) of each plasmid and hence

4uL Xtreme-GENE9. 24 hrs post-transfection, fluorescing cells were counted relative to the total number of cells in five fields of view from disparate sections of the plate under a fluorescent microscope. 48 hrs post-transfection, the cells were harvested on ice and lysed with 250uL of lysis buffer (radioimmunoprecipitation assay buffer (RIPA, Boston BioProducts), Complete Mini proteinase inhibitor tablet (Roche Applied Science), 1mM PMSF, and 5mM DTT). Lysis continued for 30 minutes at 4oC under nutation, and then tubes were spun at 10,000 g for 15 minutes at 4oC in a tabletop microcentrifuge. The supernatant was reserved, and the total protein concentration was assessed by the DC Protein System (Bio-Rad), similar to the Lowry System using BSA to make a standard curve.

Treatment to elevate intracellular cAMP levels:

For treated samples, prior to harvesting, cells were treated with 100uM 3-isobutyl-

1methylxanthine (IBMX, Sigma-Aldrich, stock solution in DMSO) and 500uM 8-(4- chlorophenylthio) adenosine 3’, 5’-cyclic monophosphate sodium (pCPT-cAMP, Sigma-Aldrich, 19

o stock solution in dH2O) in complete cell medium. HEK-293 cells were incubated at 37 C in treatment media for either 5 or 30 mins.

Co-Immunoprecipitation:

Protein lysates harvested as described above (25-500ug) were incubated with primary antibodies mouse anti-FLAG (Sigma-Aldrich, F3165, 3.8mg/mL), rabbit anti-GFP (abcam 290,

5mg/mL), or control, normal mouse or rabbit IgG (Santa Cruz 2025/2027, 2mg/mL) for 1hr.

Samples were next incubated with 20uL of protein A/G agarose beads (Santa Cruz) either for 1hr or overnight prior to spin down at 1000g (for 5 min. The pellet was washed with lysis buffer and spun down at 2500rpm for 1 min (later adjusted to 5 min for optimized protocol). This wash step was repeated four times. During these steps, the supernatant from the first and second collections from centrifugation and the pelleted beads were reserved for immunoblot analyses.

Immunoblot analysis:

Cell lysates (12-25ug) and co-IP samples (25-500ug of protein from original protein samples) were prepared with concentrated Laemmli buffer (Bio-Rad) at a final concentration of

o 1X, 1mM DTT (Sigma Aldrich), and dH2O and heated at 65 C for 30min. Protein samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5% acrylamide gels (Bio-Rad). Gels were blocked with dH2O for 15min, covered in GelCodeBlue

(Thermo Scientific Pierce) for 30-45 min, and washed five times with dH2O for a total of 30-

45min to observe all proteins on the gel. Gels were transferred to a polyvinylidene difluoride

(PVDF) membrane and then the membrane was stained with PonceauS (Sigma-Aldrich). For immunoblotting, membranes were blocked with 5% nonfat milk powder in 1X PBS. Blots were 20 incubated with primary antibodies in 5% nonfat milk in 1X PBS. Antibody dilutions were:

KvLQT1 (rabbit polyclonal, Alomone-022, 0.75mg/mL) at 1:1000 or 1:5000, hERG (rabbit polyclonal, Alomone-062, 0.6mg/mL) at 1:1000 or 1:5000, and GAPDH (mouse monoclonal,

Sigma-Aldrich G8795, 1.2mg/mL) at 1:5000 or 1:10,000. Goat anti-rabbit (abcam 97051,

1mg/mL) and goat anti-mouse (abcam 97023, 1mg/mL) horseradish peroxidase (HRP)- conjugated secondary antibodies at 1:10,000 dilution in 5% nonfat milk in 1X PBS were used to bind to the species appropriate primary antibody. Precision Plus Protein WesternC Ladder (Bio-

Rad) was detected through 1:10,000 dilution of strep tactin (Bio-Rad) in 5% nonfat milk in 1X

PBS, which was included along with the secondary antibody. Enhanced chemiluminescence

(ECL) detection reagents (Thermo Scientific Pierce) were used for imaging on a Gel Doc system

(Bio-Rad) with an exposure time of 60-150sec. Exposure time was selected based on the quality of after image.

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Results:

Representative co-immunoprecipitation:

In this thesis, the main objective is to demonstrate protein-protein interaction between hERG and KvLQT1 through the classical biochemical assay of co-immunoprecipitation (co-IP).

Non-specific IgG antibodies were utilized as negative controls to reinstate that the pulldown is specific for its intended epitope. In addition to presenting evidence of hERG-KvLQT1 interactions, the motivation behind this study is to investigate the molecular mechanisms associated with the interactions by increasing intracellular cAMP levels with membrane- permeable cAMP analogues and IBMX. Although this study does not directly delineate the role of the direct binding of cAMP to the hypothesized cyclic nucleotide homology binding domain

(cNHBD) of hERG or downstream pKA mediated effects, these experiments set up the foundation to employ co-IPs as a method to analyze these mechanisms in the near future.

Through the exposure of cAMP, it is expected that the interactions between hERG and KvLQT1 will decrease as shown through the analysis of western blotting after co-IP.

In order to determine the interactions between hERG and KvLQT1, co- immunoprecipitation (co-IP) was performed prior to immunoblot detection. Preliminary co-IPs with GFP and FLAG antibodies indicated that there are interactions between hERG and

KvLQT1 (Figure 5). GFP and FLAG tags have been previously inserted into the DNA plasmid construct of hERG and KvLQT1. More specifically, a monomeric GFP tag has been inserted at the COOH-terminus of the construct whereas the FLAG tag has been inserted at the NH2- terminus of the construct. Overall, it is assumed that these tags do not interfere with the interactions between the two proteins during the co-IP process. The same constructs have been utilized fairly consistently throughout this thesis. Additionally, a monomeric Cherry tag 22 construct, similar to that of the GFP tag, was used as complementation to constructs with the

GFP tag not only to visualize co-transfection under fluorescence but also since it has been previously identified to not interact with the GFP antibody (not shown). Generally, these epitope tags on the proteins allow for a more feasible process as antibodies for the tags can be introduced during the co-IPs and antibodies against the specific protein can be introduced during the western blotting process.

Logically, when hERG and KvLQT1 are detected after co-IP and western blotting with antibodies directed against those proteins, it was assumed that the co-IP was successful. More specifically, after a co-IP with a tagged version of hERG and wild-type KvLQT1 and if KvLQT1 was detectable after immunoblot detection, then hERG was able pulldown KvLQT1 successfully.

The same concept goes in reverse for KvLQT1 pulling down hERG. Thus, among the anti-GFP co-IPs, it seems as though hERG was able to successfully pulldown KvLQT1 and KvLQT1 was also able to pulldown hERG (Figure 5). However, within the anti-FLAG co-IP, KvLQT1 was able to pulldown hERG (Figure 5) whereas hERG was unable to pulldown KvLQT1 (not shown).

Since the FLAG-tagged hERG was unable to pulldown KvLQT1, this either indicates that hERG was simply unable to pulldown KvLQT1 successfully or that there may be a problem with the

FLAG-tagged hERG construct itself.

Moreover, when non-specific IgG antibodies were used in correspondence to the anti-

GFP co-immunoprecipitation, western blots detected non-specific bands as if pulldown was successful between two proteins found with relatively similar molecular weight as hERG and

KvLQT1 (not shown). On the other hand, western blotting detected no presence of the protein when non-specific mouse IgG antibodies introduced to the FLAG-tagged KvLQT1 and hERG

(Figure 5). As such, non-specific binding was less likely in the presence of a smaller epitope tag 23 than GFP. Although in Figure 5 these non-specific rabbit IgG antibodies were not yet used as negative controls for the anti-GFP co-IP, the following co-IPs and western blots were inclusive with non-specific IgG antibodies. These interactions using non-specific rabbit IgG antibodies were further investigated for anti-GFP co-IPs.

In lanes 3 and 7 of Figure 5, hERG and KvLQT1 continue to be present in the supernatants after the IP which indicates that the IP step is not pulling down all of the protein of interest from the cell lysates. Such results suggest that not enough antibody is binding all the protein of interest, that there are not enough beads to bind to the antibody, or that the interactions between the proteins are not strong enough to pulldown all the proteins. Ideally, complete reduction of the presence of the tagged protein in the supernatant after IP indicates that all the proteins were able to be immunoprecipitated. Since not all proteins of hERG and KvLQT1 will be interacting, it is not expected that pulldown of the corresponding protein will be absolute to the extent that there would be none left in the supernatant after IP. Moreover, to successfully semi-quantify these co-IPs, it is essential that majority, if not all, proteins interacting are pulled down. Such issues provided framework to troubleshoot the co-IP protocol by adjusting the amount of protein and corresponding antibody used during the pulldown step.

In addition, we are not consistently detecting two bands for hERG, representing the glycosylated and unglycosylated forms of the protein (Figure 5). Since hERG is a membrane protein, it is generally accepted that the top band represents the mature, functional hERG while the bottom represents protein that is still being processed and trafficked. Not only does this issue appear among westerns after co-IPs, but also among original cell lysates that were transfected with different hERG constructs. Although this inconsistency does not directly affect the interpretation co-IPs between hERG and KvLQT1, this prompted examination of differences 24 between protein samples separated from other cellular components and the crude extract of proteins lysed from HEK-293 cells.

Ultimately, through these troubleshooting co-IPs and successful display of interaction between hERG and KvLQT1, the molecular mechanisms were scrutinized by increasing intracellular cAMP levels. Accordingly, differences between treated and untreated cells were evaluated by the amount of detection of the protein being pulled down after co-IP.

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Figure 5. Representative co-immunoprecipitations of KvLQT1 and hERG demonstrate protein-protein interactions as well as prepare framework for troubleshooting the co-IP protocol. Co-IP was performed with 300ug of HEK-293 cell lysates with 12.5ug of anti-GFP or 7.6ug of anti-FLAG, incubated with beads for 1hr. Lanes 2-5: hERG-mGFP + KvLQT1-mCherry; 2: cell lysates, 3: supernatant after IP, 4: supernatant after washing IP pellet, *5: IP. Lanes 6-9: hERG-mCherry + KvLQT1-mGFP; 6: cell lysates, 7: supernatant after IP, 8: supernatant after washing IP pellet, *9: IP. Lanes 10-13: hERG + FLAG KvLQT1; 10: cell lysates, 11: supernatant after IP, *12: IP, 13: non-specific mouse IgG control (7.6ug). Immunoblot detection: hERG & KvLQT1 1o at1:1000, 2o at 1:10,000; GAPDH 1o at 1:10,000, 2o at1:10,000.

26

FLAG-tagged hERG:

To examine the pulldown of KvLQT1 via FLAG-tagged hERG, co-IPs with various concentrations of anti-FLAG were performed using lysates from cells double, transiently transfected with KvLQT1 and FLAG-tagged hERG. Using a smaller amount of total protein lysates into the co-IP to saturate the beads during the IP process, hERG was still unable to pulldown KvLQT1 (Figure 6). Since there were no distinct problems present for anti-FLAG co-

IPs where FLAG-tagged KvLQT1 pulled down hERG, it was not expected that there would be any significant differences when changing the amount of protein. In fact, a larger fraction of the total protein lysate should have been pulled down in the same amount of antibody, which was not the case. In addition to the presence of hERG and KvLQT1, bands at ~25kDa most likely represent the remaining anti-FLAG in the supernatant that is binding to the secondary antibody

(made in the same organism—mouse) during the western blot process. On the other hand, bands of hERG and KvLQT1 were detected in supernatants prior to pulldown (Figure 6), showing that the co-IP was unsuccessful and suggesting that this hERG construct does not, in fact, include a

FLAG tag. To confirm this possibility, a western blot comparing the presence of hERG with antibodies directed against the specific protein and FLAG tag was performed. hERG and

KvLQT1 were successfully detected using antibodies specific for those proteins (Figure 7). Yet, hERG was undetectable with the FLAG antibody (Figure 7). Therefore, it seems as though the hERG construct does not include a FLAG tag, making it ineffective to use such construct in the future to show the mutual downregulation of FLAG-hERG pulling down KvLQT1. 27

Figure 6. Co-immunoprecipitation with putative FLAG-hERG construct is unable to pulldown KvLQT1. Co-IP was performed with 50ug of HEK-293 cell lysates with 3.8-7.6ug of anti-FLAG antibody, incubated with beads for 1hr. Lane 2: cell lysates, Lane *3: IP with 10ug of antibody, Lane 4: supernatant after IP, Lane *5: IP with 5ug of antibody, Lane 6: supernatant after IP, Lane 7: non-specific rabbit IgG control (7.6ug), Lane 8: supernatant after IgG pulldown, Lane 9: non-specific rabbit IgG control (3.8ug), Lane 10: supernatant after IgG pulldown, Lane 11: no antibody during pulldown, Lane 12: supernatant after no antibody pulldown. Immunoblot detection: Lanes 2-9: hERG & KvLQT1 1o at 1:5000, 2o at 1:10,000; GAPDH 1o at 1:5000, 2o at1:10,000. 28

Figure 7. Putative FLAG-hERG construct does not seem to be properly tagged with FLAG. Co-IP was performed on a total of 500ug of HEK-293 cell lysates and 1000ug of CHO cell lysates with 7.6ug of anti-FLAG, incubated with beads for 1hr. Lanes 2-5, 11-14: transfected in HEK-293 cells; 2-3, 11-12: cell lysates, *4-*5, *13-*14: IP. Lanes 6-9, 15-18: transfected in CHO cells; 6-7, 15-16: cell lysates, *8-*9, *17-*18: IP. Immunoblot detection: Lanes 2-9: hERG & KvLQT1 1o at 1:1000, 2o at 1:10,000; Lanes 11-18: FLAG 1o at 1:500, 2o at 1:10,000; GAPDH 1o at 1:10,000, 2o at1:10,000.

29

Co-immunoprecipitation troubleshooting:

Anti-GFP co-IP and non-specific IgG control:

To optimize the co-IP assay for detecting interactions between hERG and KvLQT1, adjustments were made according to the total amount of protein from cell lysates, antibody, and beads into the co-IP prior to immunoblot detection. Throughout this process, the total amount of protein into the co-IP was reduced from the initial 300ug to 100ug and 50ug. The amount of antibody ranged from 10ug and 5ug as well as beads from a total resuspended volume of the recommended 20uL and 40uL. Despite these changes, there were no westerns that were conclusive enough to set a specific protocol for the anti-GFP co-IP (Figures 8-10). In particular, lanes with the anti-GFP co-IP were of similar pattern to IPs with the non-specific rabbit IgG control antibody (Figure 8). A new antibody (from abcam; the original from Santa Cruz) was purchased to see if such results were caused by non-specific binding due to the non-specific IgG antibody itself. However, there seems to be no drastic difference in the pattern where the lanes are still fairly prominently detected (Figures 9 and 10). These issues demonstrate that that there may be problems with the beads during the co-IP process or in large possibly that fluorescent tagged proteins cannot be used to analyze interactions through co-IPs unless another control is used aside from non-specific IgG antibodies.

In addition to these concerns, the co-IPs themselves are not convincing to state that the bands detected are the proteins of interest. The bands are not as detectable as lanes only with the remaining supernatant with hERG and KvLQT1 (Figures 8-10). In fact, these lanes should have been reduced so that these proteins could no longer been seen. Therefore, it was thought that changes in incubation time with the beads may have altered the pulldown process. Although standard protocols normally allow for incubation time with beads for 1hr, the company of 30 purchase (Santa Cruz) had provided a protocol where bead incubation was set for overnight.

Accordingly, co-IPs were set back to the original amount of 300ug and 12.5ug of antibody to compare the standards of bead incubation. It must also be noted that centrifugation was extended between washes during these co-IPs to ensure that all beads with proteins were properly being pulled down. Set side by side with anti-FLAG co-IPs, it seems as though anti-GFP co-IPs require overnight bead incubation (Figure 11). The anti-GFP co-IPs with 1hr bead incubation show how

KvLQT1 was unable to pulldown hERG as effectively as the overnight bead incubation (Figure

11). On the other hand, anti-FLAG co-IPs require 1hr bead incubation as the protein bands of interest are dense, making it difficult to interpret (Figure 11). A curious connection between the anti-FLAG co-IPs with overnight bead incubation and IPs with non-specific IgG control antibody is that the pattern of lane smearing seems relatively similar.

Although issues seem to be unresolved, it seems as though anti-GFP co-IPs will continue to be difficult to troubleshoot. In particular, IP with non-specific IgG control antibodies leave smears that are hard to overcome without pinpointing the problem. Moreover, KvLQT1-mGFP was not properly detected in cell lysates (Figures 8-11), making it difficult to continue to troubleshoot beyond comparisons with bead incubation. However, anti-FLAG co-IPs were successfully optimized such that pulldown can be repeatedly detected with 300ug and 10ug of anti-FLAG as KvLQT1 is no longer detectable in the supernatant (Figure 11). Thus, anti-FLAG co-IPs present their specificity and consistency in protein-protein interaction between hERG and

KvLQT1 when compared with anti-GFP co-IPs. 31

Figure 8. Representative co-immunoprecipitation with GFP tag inconclusively demonstrates interactions as co-IP lanes look similar to lanes as IP with non-specific IgG antibody. Co-IP was performed with various amounts of HEK-293 cell lysates and anti-GFP/ non-specific IgG antibody, incubated with beads for 1hr. Lane 2: cell lysates, Lane *3: IP with 100ug of protein and 10ug of anti-GFP, Lane 4: supernatant after IP, Lane *5: IP with 50ug of protein and 10ug of anti-GFP, Lane 6: supernatant after IP, Lane *7: IP with 50ug of protein and 5ug of anti-GFP, Lane 8: supernatant after IP, Lane *9: IP with 100ug of protein, 10ug of anti-GFP, and 40uL (2X normal) amount of beads, Lane 10: supernatant after IP, Lane 11: non-specific rabbit IgG control 100ug of protein and 10ug of antibody, Lane 12: supernatant after IgG pulldown, Lane 13: non-specific rabbit IgG control with 50ug of protein and 10ug of antibody, Lane 14: supernatant after IgG pulldown, Lane 15, 17: no antibody during pulldown, Lane 16,18: supernatant after no antibody pulldown. Immunoblot detection: hERG & KvLQT1 1o at1:5000, 2o at 1:10,000; GAPDH 1o at 1:5000, 2o at1:10,000. 32

Figure 9. Co-immunoprecipitation with GFP tag inconclusively demonstrates interactions between hERG and KvLQT1 as protein bands of interest are difficult to detect. Co-IP was performed with 50ug of HEK-293 cell lysates and 5ug anti –GFP/new non-specific IgG antibody, incubated with beads for 1hr. Lanes 2-9: hERG-mCherry + KVLQT1-mcherry; 2: cell lysates, *3: IP, 4: supernatant after IP, 5: IP with non-specific IgG antibody, 6: supernatant after IgG pulldown, 7: no antibody during pulldown, 8: supernatant after no antibody pulldown, 9: cell lysates. Lanes 10-17: hERG-mGFP + KVLQT1-mcherry: *10: IP, 11: supernatant after IP, 12: IP with non-specific IgG antibody, 13: supernatant after IgG pulldown, 14: no antibody during pulldown, 15: supernatant after no antibody pulldown, 16: cell lysates, 17: cell lysates. Immunoblot detection: hERG & KvLQT1 1o at1:5000, 2o at 1:10,000; GAPDH 1o at 1:5000, 2o at1:10,000.

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Figure 10. Co-immunoprecipitation with GFP tag inconclusively demonstrates interactions between hERG and KvLQT1 as protein bands of interest are difficult to detect (Western of Figure 9 redone). Co-IP was performed with 50ug of HEK-293 cell lysates and 5ug anti-GFP/new non-specific IgG antibody, incubated with beads for 1hr. Lanes 2-8: hERG-mCherry + KVLQT1-mcherry; 2: cell lysates, *3: IP, 4: supernatant after IP, 5: IP with non-specific IgG antibody, 6: supernatant after IgG pulldown, 7: no antibody during pulldown, 8: supernatant after no antibody pulldown. Lanes 9-15: hERG-mGFP + KVLQT1-mcherry: *9: IP, 10: supernatant after IP, 11: IP with non- specific IgG antibody, 12: supernatant after IgG pulldown, 13: no antibody during pulldown, 14: supernatant after no antibody pulldown, 15: cell lysates. Immunoblot detection: hERG & KvLQT1 1o at1:5000, 2o at 1:10,000; GAPDH 1o at 1:5000, 2o at1:10,000.

34

Figure 11. Co-immunoprecipitation with differences in bead incubation time changes the presence of interactions between hERG and KvLQT1. Co-IP was performed with 300ug of HEK-293 cell lysates and 12.5ug anti-GFP or 7.6ug of anti- FLAG, incubated with beads for either 1hr or overnight. Lanes 2-8: hERG-mcherry + KvLQT1-mGFP; 2: cell lysates, *3-*4: IP after 1hr incubation with beads, 5: supernatant after IP, *6-*7: IP after overnight incubation with beads, 8: supernatant after IP. Lanes 9-15: hERG + FLAG-KvLQT1: 9: cell lysates, *10-*11: IP after 1hr incubation with beads, 12: supernatant after IP, *13-*14: IP after overnight incubation with beads, 15: supernatant after IP. Immunoblot detection: hERG & KvLQT1 1o at1:5000, 2o at 1:10,000; GAPDH 1o at 1:5000, 2o at1:10,000.

35

Use of GelCode Blue and PonceauS Staining with co-IP analysis:

In order to further investigate anti-GFP co-IPs beyond immunoblot detection analysis,

GelCode Blue staining and PonceauS staining before and after transfer, respectively, were examined with the possibility that the stain would be able to specifically detect the proteins, hERG and KvLQT1. Such staining was not to particularly address the problems directly, but rather to observe whether these proteins could be detected without worry of non-specific binding with the primary and secondary antibodies during the western. In particular, it seemed possible that the non-specific rabbit IgG control antibody was somehow interacting with the secondary antibody of goat-anti-rabbit that normally targets against anti-hERG and anti-KvLQT1 primary antibodies. However, GelCode Blue and PonceauS staining were unable to detect any proteins besides the heavy and light chains of anti-GFP (Figures 12 & 13). When the gel was stained with

GelCode Blue, differences between the supernatants after IP via the anti-GFP and the supernatants after IP via the non-specific IgG control seem to be set apart by the heavy and light chains of the anti-GFP antibody (Figure 12). There seems to be a few bands at about ~50kDa in the co-IPs and IPs with the non-specific IgG antibody, but these bands are not specifically bands representing either hERG or KvLQT1. Other staining with membranes also showed similar patterns that could not discern the proteins of interest. This is most likely because these staining are not specific and sensitive enough to detect either hERG or KvLQT1.

Although GelCode Blue staining seems to be more capable than PonceauS staining to detect any changes, transfer was unsuccessful when gels were stained with GelCode Blue. It was later found that it would take three times longer (standard Bio-Rad protocol) to transfer gels after such staining; therefore, GelCode Blue staining was reserved for western blot analysis where it was necessary to see if proteins were present. Since GelCode Blue staining was not sensitive 36 enough to detect either hERG or KvLQT1, this step was ultimately removed from these protocols.

Figure 12. GelCode Blue staining is unable to detect specific proteins of interests after co- immunoprecipitation of hERG-mCherry + KvLQT1-mGFP (Western—Figure 8). Co-IP was performed with various amounts of HEK-293 cell lysates and anti-GFP/ non-specific IgG antibody, incubated with beads for 1hr. Lane 2: cell lysates, Lane *3: IP with 100ug of protein and 10ug of anti-GFP Lane 4: supernatant after IP, Lane *5: IP with 50ug of protein and 10ug of anti-GFP, Lane 6: supernatant after IP, Lane *7: IP with 50ug of protein and 5ug of anti-GFP, Lane 8: supernatant after IP, Lane *9: IP with 100ug of protein, 10ug of anti-GFP, and 40uL (2X normal) amount of beads, Lane 10: supernatant after IP, Lane 11: IP with non-specific rabbit IgG control 100ug of protein and 10ug of antibody, Lane 12: supernatant after IgG pulldown, Lane 13: non-specific rabbit IgG control with 50ug of protein and 10ug of antibody, Lane 14: supernatant after IgG pulldown, Lane 15: no antibody during pulldown, Lane 16: supernatant after no antibody pulldown, 17: cell lysates.

37

Figure 13. PonceauS staining after transfer is unable to detect specific proteins of interest after co-immunoprecipitation of hERG-mCherry + KvLQT1-mGFP (Western—Figure 8). Co-IP was performed with various amounts of HEK-293 cell lysates and anti-GFP/ non-specific IgG antibody, incubated with beads overnight.

38

Crude extracts vs. supernatant after harvesting cells:

In order to examine the proteins more closely, crude extracts from harvested cells were compared to supernatant taken after the centrifugation step during the normal harvesting protocol.

More specifically, this comparison was made due to the possibility that important proteins or proteins found at the membrane may have been discarded after cell lysis. Previous westerns have shown that depending on the different construct of hERG, not all the hERG proteins were detected by the expected two bands suggesting that certain hERG constructs allowed for better trafficking to the cell membrane (not shown). In such case, these properly trafficked hERG proteins that were membrane-bound may have been discarded after cell lysis. Immunoblot detection showed that there was no change in the higher molecular weight band of hERG (Figure

14). In fact, it seems as though the more saturated band is the glycosylated form of hERG, as the top band present with the hERG-mCherry construct is in closer proximity to the band present with the hERG-mGFP construct (Figure 14). In particular, the hERG-mCherry construct normally presents two bands whereas the hERG-mGFP construct shows only one band, presumably the glycosylated form (not shown). Moreover, although the bands are slightly more saturated in crude extracts, the differences in protein expression does not suggest that a significant fraction of the proteins of interest were discarded in the pellet following the final centrifugation step of cell harvest and lysis. In fact, preliminary ImageJ quantification analysis depicted no significant differences between crude extract samples and supernatant after harvesting cells. In addition, GelCode Blue staining before western blotting showed that there were no qualitative differences between crude extracts and supernatant collections (Figure 15).

Bands seemed slightly fainter among supernatant lanes; however, there were no signs of changes 39 in the pattern of proteins (Figure 15). Therefore, the original protocol to take the supernatant after harvesting cells was maintained.

Figure 14. Detection of protein bands between crude extracts of cell lysates is not significantly different to supernatant collection after spin-down. Crude extract and supernatant samples are from harvesting and lysing HEK-293 cells. Even numbers: supernatant collection, odd numbers: crude extract. Lanes 2-3: untransfected HEK-293 cell lysates, 4-5: putative FLAG hERG + KVLQT1-S1-myc- S2, 6-7: hERG + FLAG-KvLQT1, 8-9: hERG-mGFP + KVLQT1-mCherry, 10-11: hERG- mCherry + KVLQT1-mGFP. Immunoblot detection: hERG & KvLQT1 1o at1:5000, 2o at 1:10,000; GAPDH 1o at 1:5000, 2o at1:10,000. ImageJ quantification was used to determine band density and showed no significant difference between each set of bands.

40

Figure 15. GelCode Blue staining demonstrates that crude extracts of cell lysates seem similar to supernatant collection after spin-down (corresponding western-Figure10). Crude extract and supernatant samples are from harvesting and lysing HEK-293 cells. Even numbers: supernatant collection, odd numbers: crude extract. Lanes 2-3: untransfected HEK-293 cell lysates, 4-5: putative FLAG hERG + KVLQT1- S1-myc- S2, 6-7: hERG + FLAG-KvLQT1, 8-9: hERG-mGFP + KVLQT1-mCherry, 10-11: hERG- mCherry + KVLQT1-mGFP.

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Treatment with IBMX+pCPT-cAMP vs. no treatment:

In order to determine the role of the cNBHD of hERG in the interactions between hERG and KvLQT1, intracellular cAMP levels were increased by treatment with IBMX and pCPT- cAMP (a membrane-permeable cAMP analog). HEK-293 cells were treated with IBMX and pCPT-cAMP by bath application in medium for 30 min. prior to harvesting. KvLQT1 was able to successfully pulldown hERG in treated and untreated cells in both anti-GFP and anti-FLAG co-IPs (Figure 16). Although, qualitatively, KvLQT1 was able to pulldown more hERG in untreated cells using anti-GFP for co-IP (Figure 16), the anti-FLAG co-IP seemed to suggest that this was not the case. Yet, it is difficult to distinguish whether the pulldown suggests such an argument as the relationship of pulldown does not clearly exemplify if hERG is truly pulling down more KvLQT1 in untreated cells (Figure 16). Additionally, in lane *15, it there seems to be an oversaturation of hERG after co-IP without treatment rather than reduced pulldown.

Another issue with Figure 16 is that the presence of GAPDH was almost undetectable. Since the protocol for detecting GAPDH developed utilizing Chinese Hamster Ovary (CHO) cells rather than HEK-293 cells, perhaps detection of the proteins of interest was overly prominent, and

GAPDH detection was reduced. Therefore, changes in 1o antibody ratio GAPDH was altered for all western blot protocols from 1:50,000 to 1:5000. Since this necessary control was initially unsuccessful, another western blot was necessary to further establish the relationship between treated and untreated cells (Figure 17). Additional qualitative analysis of lanes *8 and *14

(untreated) compared to lanes *4 and *12 (treated) suggests that hERG was able to pulldown more KvLQT1 when untreated (Figure 17). Although no differences were seen in protein detection through GelCode Blue staining (Figure 18), western blotting is more demonstrative of the presence of the specific proteins of interest. Therefore, this implies that increase in 42 intracellular cAMP levels disrupts the interactions between the proteins, hERG and KvLQT1, at the biochemical level.

In addition, co-IPs with a smaller protein input and shorter treatment were performed to consider if differences in interactions could still be seen. With a smaller protein input, the anti-

FLAG co-IPs suggest that differences between the detection of hERG were less prominent

(Figure 19). Although lanes *9 and *10 (treated cells) suggest that KvLQT1 was unable to pulldown as much hERG as lanes *4 and *5, the differences were not as evident as earlier in

Figure 17 and KvLQT1 could not be properly detected (Figure 19). Hence, the relationship between the two proteins was more ambiguous. Moreover, when comparing co-IPs across untreated, treated cells for 5 min, and treated cells for 30 min, it was difficult to qualitatively assess any differences (Figure 20). However, this may be explained by the smaller protein input to which differences are less outstanding. Accordingly, results comparing the changes in protein- protein interactions through co-IPs should continue to be considered as inconclusive until experiments can be repeated multiple times.

As troubleshooting have yet to be refined using anti-GFP co-IPs, comparative analysis of treatment with intracellular cAMP levels also have yet to be completed. Even if studies can proceed with anti-FLAG co-IPs, the putative FLAG-tagged hERG construct needs to be re- sequenced and established to complement FLAG-KvLQT1 co-IPs. Bead incubation with anti-

GFP and anti-FLAG co-IPs have been established while amount of protein and antibody input in co-IPs need further investigation. Other additional changes in protocols that require the use of crude extract after cell lysis and the use of GelCode Blue staining as well as PonceauS staining are not necessary, and henceforth been removed from the existing protocol. Overall, as soon as optimization of co-IPs have been found, a semi-quantitative analysis of western blots will prove 43 to be a powerful tool to investigate the molecular mechanisms modulating the interactions between hERG and KvLQT1.

Figure 16. Co-immunoprecipitations of KvLQT1 and hERG after acute treatment (30 min.) with 500µM pCPT-cAMP + 100µM IBMX do not conclusively demonstrate differences in protein-protein interactions. Co-IP was performed on a total of 300ug of HEK-293 cell lysates with 12.5ug of anti-GFP or 7.6ug of anti-FLAG, incubated with beads overnight. Lanes 1-4: hERG-mCherry + KvLQT1-mGFP with IBMX + pCPT-cAMP; 1: cell lysates, 2: supernatant after IP, *3: IP, 4: non-specific rabbit IgG control. Lanes 5-8: hERG-mCherry + KvLQT1-mGFP; 5: cell lysates, 6: supernatant after IP, *7: IP, 8: non-specific rabbit IgG control. Lanes 9-12: hERG + FLAG KvLQT1 with IBMX + pCPT-cAMP; 9: cell lysates, 10: supernatant after IP, *11: IP, 12: non-specific mouse IgG control. Lanes 13-16: hERG + FLAG KvLQT1; 13: cell lysates, 14: supernatant after IP, *15: IP, 16: non-specific mouse IgG control. Immunoblot detection: hERG & KvLQT1 1o at1:1000, 2o at1:10,000; GAPDH 1o at 1:10,000, 2o at1:10,000. 44

Figure 17. Co-immunoprecipitations of KvLQT1 and hERG after acute treatment (30 min.) with 500µM pCPT-cAMP + 100µM IBMX suggests differences in protein-protein interactions. Co-IP was performed on a total of 300ug of HEK-293 cell lysates with 12.5ug of anti-GFP or 7.6ug of anti-FLAG, incubated with beads overnight. Lanes 2-5: hERG-mCherry + KvLQT1-mGFP with IBMX + pCPT-cAMP; 2: cell lysates, 3: supernatant after IP, *4: IP, 5: non-specific rabbit IgG control. Lanes 6-9: hERG-mCherry + KvLQT1-mGFP; 6: cell lysates, 7: supernatant after IP, *8: IP, 9: non-specific rabbit IgG control. Lanes 10-13: hERG + FLAG KvLQT1 with IBMX + pCPT-cAMP; 10: cell lysates, 11: supernatant after IP, *12: IP, 13: non-specific mouse IgG control. Lanes 14-17: hERG + FLAG KvLQT1; 14: IP, 15: supernatant after IP, *16: cell lysates, 17: non-specific mouse IgG control. Immunoblot detection: hERG & KvLQT1 1o at1:5000, 2o at1:10,000; GAPDH 1o at1:5000, 2o at1:10,000. 45

Figure 18. GelCode Blue staining after acute treatment (30 min.) with 500µM pCPT-cAMP + 100µM IBMX do not provide additional information in protein-protein interactions. Co-IP was performed on a total of 300ug of HEK-293 cell lysates with 12.5ug of anti-GFP or 7.6ug of anti-FLAG, incubated with beads overnight. Lanes 2-5: hERG-mCherry + KvLQT1-mGFP with IBMX + pCPT-cAMP; 2: cell lysates, 3: supernatant after IP, *4: IP, 5: non-specific rabbit IgG control. Lanes 6-9: hERG-mCherry + KvLQT1-mGFP; 6: cell lysates, 7: supernatant after IP, *8: IP, 9: non-specific rabbit IgG control. Lanes 10-13: hERG + FLAG KvLQT1 with IBMX + pCPT-cAMP; 10: cell lysates, 11: supernatant after IP, *12: IP, 13: non-specific mouse IgG control. Lanes 14-17: hERG + FLAG KvLQT1; 14: cell lysates, 15: supernatant after IP, *16: IP, 17: non-specific mouse IgG control.

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Figure 19. Co-immunoprecipitations of KvLQT1 and hERG after acute treatment (30 min.) with 500µM pCPT-cAMP + 100µM IBMX suggests differences in protein-protein interactions. Co-IP was performed on a total of 150ug of HEK-293 cell lysates with 7.6ug of anti-FLAG, incubated with beads for 1hr. Lanes 2-6: hERG + FLAG KvLQT1; 2: cell lysates, 3: supernatant after IP, *4-*5: IP, 6: non- specific mouse IgG control. Lanes 7-11: hERG + FLAG KvLQT1 with IBMX + pCPT-cAMP; 7: cell lysates, 8: supernatant after IP, *9-*10: IP, 11: non-specific mouse IgG control. Immunoblot detection: hERG & KvLQT1 1o at1:5000, 2o at1:10,000; GAPDH 1o at1:5000, 2o at1:10,000.

47

Figure 20. Co-immunoprecipitations of KvLQT1 and hERG after acute treatment (5 min. or 30 min.) with 500µM pCPT-cAMP + 100µM IBMX suggests no detectable differences in protein-protein interactions. Co-IP was performed on a total of 150ug of HEK-293 cell lysates with 7.6ug of anti-FLAG, incubated with beads for 1hr. Lanes 2-6: hERG + FLAG KvLQT1; 2: cell lysates, 3: supernatant after IP, *4-*5: IP, 6: non- specific mouse IgG control. Lanes 7-11: hERG + FLAG KvLQT1 with IBMX + pCPT-cAMP for 5 min.; 7: cell lysates, 8: supernatant after IP, *9-*10: IP, 11: non-specific mouse IgG control. Lanes 12-16: hERG + FLAG KvLQT1 with IBMX + pCPT-cAMP for 5 min.; 12: cell lysates, 13: supernatant after IP, *14-*15: IP, 16: non-specific mouse IgG control. Immunoblot detection: hERG & KvLQT1 1o at1:5000, 2o at1:10,000; GAPDH 1o at1:5000, 2o at1:10,000.

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Discussion and Future Directions:

The goal of this study was to optimize the classical biochemical assay of co- immunoprecipitation (co-IP) to best detect protein-protein interaction between hERG and

KvLQT1. To recapitulate experiments demonstrating these protein-protein interactions between hERG and KvLQT1, co-IPs were performed under various circumstances to optimize our protocol and then use this assay to determine whether elevated levels of cAMP cause a decrease in interactions between hERG and KvLQT1. Furthermore, the incentive for this study was to examine the molecular mechanism modulating the interactions by increasing intracellular levels of cAMP. While this thesis does not directly answer questions to determine the role of the direct binding of cAMP to the hypothesized cyclic nucleotide homology binding domain (cNHBD) of hERG or downstream pKA mediated effects, the results demonstrated in this thesis provide strong evidence that co-IPs can be used a method to analyze these mechanisms in the near future.

One of the future goals is to demonstrate interactions through semi-quantitative measures; yet, troubleshooting and optimization of the co-IP procedure remain. hERG was shown to be successfully pulled by KvLQT1 using a FLAG tag (Figure 5). Results have yet to demonstrate that hERG is also able to pulldown KvLQT1 using a FLAG tag; however, the FLAG-tagged hERG construct itself must be re-sequenced and then potentially re-constructed before further analysis (Figures 6 and 7). Additionally, although hERG and KvLQT1 seem to be successfully interacting with one another (Figure 5), non-specific IgG controls showed some ability to bind non-specifically to bands similar to the proteins of interest (Figures 8-10). In such case, troubleshooting is still necessary to reduce the presence of other bands when imaging immunoblot membranes. Futher work is also required to saturate beads with the proteins to pulldown as much protein as possible. Specifically, components such as the amount of protein, 49 antibody, bead input in the co-IP have not be optimized to the level in which future experiments can consistently show successful pulldown of the proteins.

However, in this thesis, bead incubation, centrifugation timing, possible use of staining, investigation of crude extracts of cell lysates, and loading controls have been properly established into the standard protocol. In particular, anti-GFP co-IPs need a total overnight bead incubation time whereas 1hr is appropriate for anti-FLAG co-IPs (Figure 11). Centrifugation was extended between washes of the co-IP to ensure that all beads were pelleted for proper pulldown of proteins. Staining with GelCode Blue and PonceauS did not provide as much information as proteins of interest could not be specifically detected (Figures 12 and 13). GelCode Blue staining did provide more information than PonceauS staining; yet, GelCode Blue staining made the transfer process of the gel to membrane more complex. Thus, these staining methods have been reserved when information is direly necessary to assess all proteins of interest. In addition, there was no drastic difference in protein detection levels or product in crude extracts of the cell lysates (Figure 14), and therefore work continued using the supernatant of whole cell lysates. As for GAPDH presence, the 1o antibody concentration was increased to detect bands for all protein samples obtained from HEK-293 cells.

Due to the inconsistencies in our co-IPs and western blots, during future investigations, the protocol needs to be specifically reviewed. Factors that may influence the outcome of co-IPs include: different DNA constructs/plasmids, transfection efficiency, cell growth during the transfection process, cell lysis and harvesting, proper washing techniques during co-IPs, antibody and bead concentration, and product changes in beads and antibodies. Fortunately, anti-FLAG co-IPs does not present the same problems as anti-GFP co-IPs as beads seem to be saturated with the provided amount of antibody and nonspecific IgG controls detect no proteins. Therefore, if 50 protocols can be adjusted to simply minimize the amount of antibody, then the standard protocol for anti-FLAG co-IPs is very close to well-established. Overall, a general, standard protocol has been set for future co-IPs in the Darling Lab that would interpret interactions between the proteins as efficiently as possible.

These results are in-line with those from previous studies showing interactions between hERG and KvLQT1 are direct and specific using both surface plasma resonance and co-IPs, respectively (25). Results from surface plasma resonance have established the relationship between hERG and KvLQT1 to be due to the direct, physical interaction between the COOH- termini of KvLQT1 and hERG (25). To assay for the physical interactions, fragments of hERG was immobilized on a sensor surface and KvLQT1 fragments were flowed across in various concentrations (25). A strong response was detected to which indicated a physical association between the two proteins (25). Furthermore, in addition to non-specific IgG controls, it has been demonstrated that the interaction between hERG and KvLQT1 are specific using a pseudonegative control of protein, Kir2.1 (25). Since Kir2.1 was not expected to functionally interplay with the proteins of interest, unsuccessful pulldown during the co-IP process confirmed that hERG and KvLQT1 were more likely to be specifically interacting with one another (25).

According to these definitive results, the Darling Lab continues to note that these interactions are specific and direct. Although the lab has not definitively re-established such statements through own experiments, we use these terms of direct and specific interactions as the standard when demonstrating protein-protein interactions between hERG and KvLQT1.

A particularly noteworthy part of this thesis is how the interactions between hERG and

KvLQT1 exist among fluorescently-tagged proteins. Although previous studies have shown that the interactions are specific and direct (25), they primarily used other epitope tags such as HA 51 and FLAG. hERG and KvLQT1 with small tags can therefore be recapitulated with large, fluorescent protein tags using co-IPs. While there may have been some problems with negative controls to definitively state that the interactions can be successfully shown through co-IPs, the interactions are in fact present. As such, the genetic addition of the fluorescent tag does not affect the basic interactions between the proteins, as assessed through co-IPs. Accordingly, when the lab has been using these fluorescent tags to assess interactions in apFRET and electrophysiology experiments, it can be stated that these tags should not alter the integrity of the proteins of interest.

As protocols for co-IPs are not yet optimized, quantification of co-IPs has not been completed. Although previous studies have quantified westerns have based on the density of the protein bands, quantification of immunoblots do not directly relay information about how much protein is actually present. However, the ratio of densiometry of proteins provides data across lanes of samples. Therefore, when co-IPs are consistent, semi-quantification will be a powerful tool to examin the relationship of the amount of protein pulled down by the other. In particular, the differences between wild-type and altered cells (whether they may be treated with IBMX + pCPT-cAMP, altered with phosphomimetic or phosphonull mutants) can be detected with supportive numbers rather than qualitative observation. Despite such positive possibilities, issues of reliability will arise due to the lack of sensitivity of co-IPs and inconsistency of the westerns.

That said, these biochemical assays are mainly present to complement other quantitative analyses, such as apFRET and electrophysiology.

Although this study focuses primarily on the interactions of the alpha-subunits of hERG and KvLQT1, the mechanism of beta-subunit KCNE1 within the interactions has yet to be analyzed. While hERG does not have a defined role for its proposed beta-regulatory subunit(s), 52

KvLQT1 assembles with its beta-subunit, minK, to produce the proper IKs current (34). Previous studies have not yet investigated the changes in interactions due to the addition of the minK subunit on KvLQT1 (25). Though there is no indication that there would be drastic changes in interaction between hERG and KvLQT1, the beta-subunit cannot be ignored. In fact, the interactions between the alpha-subunits are not completely representative of proper interactions between the two proteins in their functional (in vivo) states. Accordingly, stable cell lines are currently being established with fluorescently labeled hERG and minK-KvLQT1 constructs.

Stable cell lines will provide protein products that express the proteins of interest consistently, allowing transfection efficiency to be less of a potentially confounding variable. However, an issue with stable cell lines is its maintainability compared to that of transient transfections.

Moreover, the combined molecular weight of a minK-KvLQT1construct is similar to that of hERG would make it difficult to analyze as one sample. To study these proteins at the functional level, electrophysiology research (Cameron Lab) has also been conducted to complement these biochemical assays and apFRET experiments in the Darling Lab.

To determine the effect of increased intracellular cAMP levels on the interactions between hERG and KvLQT1, treatment with IBMX and pCPT-cAMP was induced to cells for

30min. Preliminary results are inconclusive in ascertaining that the interactions are disrupted by the presence of cAMP; however, it is suggested that the interactions between hERG and

KvLQT1 decreased with increased levels of cAMP (Figures 17, 19). Following westerns suggest that changes in protein input may make it more difficult to determine such disrupted interactions

(Figures 19, 20). Yet, this does not deny that interactions are less prominent with treatment. In this case where there is evidence for less interaction between the two proteins, they support prior and ongoing studies that also present similar, quantitative results through apFRET (18, 22). In 53 complementation to apFRET experiments, these biochemical assays with co-IPs provide further evidence to reflect the hypothesized reduction in interactions between hERG and KvLQT1 following elevation of cAMP levels.

During apFRET experiments, cells have been treated for 5 min where changes in interactions are observable (18, 22). Treatments have been extended to 30 minutes and into hours to observe the effects of longer exposure. When initially considering the sensitivity of co-IPs and detection through immunoblotting, 30 min exposure was chosen as an alternative to 5 min treatment for biochemical assays. Accordingly, treatment of cells at shorter periods has to be revisited to ascertain whether this phenomenon of decreased interactions is observable at those time points. In addition to replicating co-IPs between treated and untreated scenarios with the most updated protocol, future studies should look into prolongation of treatment. It has been suggested that the interactions will not only offer more understanding to the role of the cNBHD, but also relate to the beta-adrenergic pathway (8, 9, 36). Overall, cAMP levels increase as a result of epinephrine binding to its beta-adrenergic receptor, resulting in the activation of adenylyl cyclase and the production of cAMP (8, 9, 36).The secondary messenger then goes to activate PKA, phosphorylating hERG and other protein targets including KvLQT1 (8, 9, 36).

Accordingly, the direct functionality of prolonging cAMP levels to the beta-adrenergic signaling further extends our understanding of permanent cardiac diseased states.

Over the duration of this study, recent publication has shifted the perception in the field regarding the potential role of the hERG cNBHD. While it has been proposed that cAMP binds to the cNBHD of hERG (8, 9) , researchers have argued that the cNBHD does not have the chemical and physical properties to bind cAMP (4, 15, 20). By crystallizing the protein channel, it was shown that the structure of the cNBHD cannot bind cAMP. In addition, fluorescence and 54 electrophysiological methods indicated that cyclic nucleotides bind with low affinity to the isolated cNBHD of hERG channels (5). Interestingly, it has been proposed that cAMP also had no effect on the current from hERG channels even at high concentrations (5). Accordingly, the

Darling Lab has shifted the focus to delineating the role of the cNBHD and downstream PKA mediated effects. Previous studies have suggested that PKA phosphorylation of hERG mediates the rate of channel synthesis as a result of elevated intracellular cAMP levels (8, 9, 36). It has been proposed that direct phosphorylation of the channel protein by PKA is involved in the regulation of hERG channel activation, and the PKA-mediated part of the shift in the hERG activation curve can be abolished by mutating all PKA-specific phosphorylation sites in the hERG channel protein (20). Therefore, phosphonull and phosphomimetic mutants of hERG have been produced (Amanda Papakryikos ’14; Medeea Popescu’17) to address the biological question of the role of PKA phosphorylation of hERG modulating hERG-KvLQT1 interactions.

In addition to co-IPs, biotinylation-based surface expression assays is another biochemical experimental component that may also shed light on the interactions between hERG and KvLQT1. Generally, these biotinylation assays can be used to assess the glycosylation of hERG to delineate the presence of the mature and immature form of hERG (8, 9). More specficially, they allow the separation of surface expressed (maturely glycosylated) hERG from that still involved in trafficking or recycling. In the Darling Lab, these biotinylation assays may also address whether the proteins of interest are properly trafficked to the membrane. Although biotinylation does not specifically represent the interactions between hERG and KvLQT1, it is expected that there would be differences upon long-term treatment (hours to days) as trafficking of hERG would be disrupted (8, 9). Accordingly, there would be discrepancy in the maturation of hERG between treated and untreated cells, as well as potentially in various exposure times of 55 elevated cAMP levels. In fact, other labs interested in cAMP effects on channel function have shown differences in surface expression with acute (<1hr) versus long-term (hours to days) treatment (8). When treated with IBMX and pCPT-cAMP over a long period of time, sustained levels of cAMP effectively increased the functional expression of hERG channel current (8).

Other studies have proposed the idea that that through the prolonged exposure to cAMP increases in hERG expression, effectively rescuing the mutual, functional downregulation (8, preliminary studies shown in McDonald lab). In such case, we can potentially assess these changes by detecting different levels of expression between the mature, glycosylated form and the immature, trafficking form of hERG through western blot analyses. Although the bands may be more difficult to distinguish, these mature and immature bands of hERG may also be quantified similarly to the co-IPs.

Current results demonstrate that increased levels of cAMP somehow interfere with the interaction between hERG and KvLQT1. Further research is necessary to delineate the molecular mechanisms associated with cAMP to which may be caused by the binding to the cNBHD or downstream, PKA-mediated effects. Ultimately, through the complete optimization of co-IPs, particularly of anti-GFP co-IPs, our studies can examine the mechanism(s) of hERG-KvLQT1 interactions through biochemical assays. Despite inconclusive results, optimization of co-IPs and preliminary results potentially furthers our understanding of the physiological regulation of hERG-KvLQT1 interactions and its implications on cardiac arrhythmias in both healthy and diseased states, as well as characterize novel interactions between two distinct potassium channel families.

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References:

1. Abbott GW, Xu X, Roepke TK. Impact of ancillary subunits on ventricular repolarization. J. Electrocardiol. 2007; 40:S42–46.

2. Anantharam A and Abbott GW. Does hERG coassemble with a beta subunit? Evidence for roles of MinK and MiRP1. Novartis Found Symp. 2005; 266: 100-112.

3. Baldi L, Hacker DL, Adam M, and Wurm FM. Recombinant protein production by large- scale transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnol. Lett. 2007; 29: 677-684.

4. Bollin F, Dechavanne V, and Chevalet L. Design of Experiment in CHO and HEK transient transfection condition optimization. Protein Expression and Purification. 2011 July; 78(1): 61-68.

5. Brelidze TI, Carlson AE, Zagotta WN. Absence of direct cyclic nucleotide modulation of mEAG1 and hERG1 channels revealed with fluorescence and electrophysiological methods. J. Biol Chem. 2009 Oct; 284(41): 27989-27997.

6. Bredlidze TI and Zagotta WN. Exploring Ligand Regulation of Ion Channels in the EAG Family. Biophysical Society. 2011; 100(3): p103a.

7. Brunner M, Peng X, Liu GX, Ren XQ, Ziv O, Choi BR, Mathur R, Hajjiri M, Odening KE, Steinberg E, Folco EJ, Pringa E, Centracchio J, Macharzina RR, Donahay T, Schofield L, Rana N, Kirk M, Mitchell GF, Poppas A, Zehender M, Koren G. Journal of Clinical Investigation. 2008 Jun; 118(6): 2246-2259. Pub 2008 May 08.

8. Chen J, Sroubek J, Krishnan Y, Li Y, Bian J, McDonald TV. PKA phosphorylation of HERG protein regulates the rate of channel synthesis. American Journal of Physiology- Heart and Circulatory Physiology. 2009 Feb; 296: H1244-H1254. Pub 2012 Aug 30.

9. Cui J, Melman Y, Palma E, Fishman GI, McDonald TV. Cyclic AMP regulates the HERG K+ channel by dual pathways. Current Biology. 2000 April; 10: 671-674.

10. Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell. 1995; 80: 795–803.

11. Ehrlich, JR, Pourrier M, Weerapura M, Ethier N, Marmabachi AM, Hebert TE, Nattel S. KvLQT1 modulates the distribution and biophysical properties of HERG: A novel a- subunit interaction between delayed rectifier currents. The Journal of Biological Chemistry. 2004 Jan; 279: 1233-1241.

12. Finley MR, Li Y, Hua F, Lillich J, Mitchell KE, Ganta S, Gilmour Jr. RF, Freeman LC. Expression and coassociation of ERG1, KCNQ1, and KCNE1 potassium channel proteins 57

in horse heart. American Journal of Physiology-Heart and Circulatory Physiology. 2002 July; 283: H126-H138.

13. Goldman AM, Glasscock E, Yoo J, Chen TT, Klassen TL, and Noebels JL. Arrhythmia in heart and brain: KCNQ1 mutations link epilepsy and sudden unexplained death. Sci Transl Med. 2009 Oct; 1(2): 2ra6.

14. Gong Q, Anderson CL, January CT, and Zhou Z. Role of glycosylation in cell surface expression and stability of HERG potassium channels. American Journal of Physiology- Heart and Circulatory Physiology. 2002 July; 283(1): H77-H83.

15. Haitin H, Carlson AE, and Zagotta WN. The structural mechanism of KCNH-channel regulation by the eag domain. Nature. 2013 Sept; 501: 444-448.

16. Jackson H and Accili EA. Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations. BMC Evolutionary Biology. 2008; 8: 188.

17. Jespersen T, Grunnet M, and Olesen SP. The KCNQ1 Potassium Channel: From Gene to Physiological Function. American Physiological Society: Physiology. 2005 Dec; 20(6): 408-416.

18. Lee YL, Kim E, Organ-Darling LE. Examining the role of direct cAMP-binding bersus PKA-mediated effects on interactions between the cardiac potassium channel alpha- subunit proteins hERG and KvLQT1. Biophysical Society Journal. 2015

19. Levin G, Chikvashvili, D, Singer-Lahat D, Peretz T, Thornhill WB, and Lotan I. Phosphorylation of a K+ channel alpha subunit modulates the inactivation conferred by a beta subunit. Involvement of cytoskeleton. J Biol Chem. 1996 Nov; 271(46): 29231- 29328.

20. Li Q, Wong YL, Ng HQ, Gayen S, and Kang CB. Structural insight into the transmembrane segments 3 and 4 of the hERG potassium channel. Journal of Peptide Science. 2014 Oct; 20 (12): 935-944.

21. Noble D and Tsien RW. Outward membrane currents activated in the plateau range of potentials in cardiac Purkinje fibres. The Journal of Physiology. 1969 Jan; 200(1): 205- 231.

22. Organ-Darling LE, Vernon AN, Giovaniello JR, Lu Y, Moshal K, Roder K, Li W, Koren G. Interactions between hERG and KCNQ1 α-subunits are mediated by their COOH termini and modulated by cAMP. American Journal of Physiology-Heart and Circulatory Physiology. 2013 Feb; 304: H589-599. Pub 2012 Dec 15.

58

23. Petrecca K, Atanasiu R, Akhavan A, Shrier A. N-linked glycosylation sites determine HERG channel surface membrane expression. The Journal of Physiology. 1999 Feb; 515: 41-48.

24. Port JD, and Bristow MR. Altered beta-adrenergic receptor gene regulation and signaling in chronic heart failure. J Mol Cell Cardiol. 2001 May; 33(5): 887-905.

25. Ren XQ, Liu GX, Organ-Darling LE, Zheng R, Roder K, Jindal HK, Centracchio J, McDonald TV, Koren G. Pore Mutants of HERG and KvLQT1 Downregulate the Reciprocal Currents in Stable Cell Lines. American Journal of Physiology-Heart and Circulatory Physiology. 2010 Nov; 299(5):H1525-34. Epub 2010 Sep 10.

26. Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-spanning receptors and heart function. Nature. 2002; 415: 206–212.

27. Roden DM, Balser JR, George AL Jr., and Anderson ME. Cardiac ion channels. Annu. Rev. Physiol. 2002; 64:431–475.

28. Roden DM. Taking the “Idio” out of “Idiosyncratic”: Predicting Torsades de Pointes. Pacing and Clinical Electrophysiology. 1998 May; 21(2): 1029-1034. EPub 30 Jun 2006.

29. Sherwood, L. Human Physiology. BroksCole. 2012; 8(13).

30. Shieh CC, Coghlan M., Sullivan JP, and Gopalakrishnan M. Potassium channels: molecular defects, diseases, and therapeutic opportunities. Pharmacol. Rev. 2000; 52:557–594.

31. Snyders DJ. Structure and function of cardiac potassium channels. Cardiovasc. Res. 1999; 42:377–390.

32. Splawski I, Tristani–Firouzi M, Lehmann MH, Sanguinetti MC, and Keating MT. Mutations in the hminK gene cause long QT syndrome and suppress IKs function. Nat Genet. 1997; 17: 338–340.

33. Strutz-Seebohm N, Pusch M, Wolf S, Stoll R, Tapken D, Gerwert K et al. Structural basis of slow activation gating in the cardiac IKs channel complex. Cellular Physiology and Biochemistry. 2011; 27(5): 443-452.

34. Takumi T, Ohkubo H, and Nakanishi S. Cloning of a membrane protein that induces a slow voltage-gated potassium current. Science. 1988; 1042-1045.

35. Tristani-Firouzi M and Sanguinetti MC. Voltage-dependent inactivation of the human K+ channel KvLQT1 is eliminated by association with minimal K+ channel (minK) subunits. The Journal of Physiology. 1998; 510: 37-45.

59

36. Thomas D, Zhang W, Karle CA, Kathofer S, Schols W, Kubler W, and Kiehn J. Deletion of Protein Kinase A Phosphorylation Sites in the HERG Potassium Channel Inhibits Activation Shift by Protein Kinase A. The Journal of Biological Chemistry. 1999 Sept; 274: 27457-27462.

37. Vandenberg JI, Perry MD, Perrin MJ, Mann SA, Ke Y, Hill AP. hERG K+ Channels: Structure, Function, and Clinical Significance. American Physiological Society: Physiological Reviews. July 2012; 92(3): 1393-1478.

38. Warmke JW, Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci USA. 1994; 91: 3438–3442.

39. Zheng R, Negro WA, Rhadames AR, Laska A, Cho SI, Cheng H, Obeg-Gyimah E, McDonald T. Ligand-Binding Characteristics of the Cyclic Nucleotide Binding Domain (CNBD) of the HERG Potassium Channel and Long QT Syndrome. Biophysical Journal. 2012 Jan; 102(3): 329a.

40. Zheng W, Verlander JW, Lynch IJ, Cash M, Shao J, Stow LR, Cain BD, Weiner D, Wall SM, Wingo CS. Cellular distribution of the potassium channel KCNQ1 in normal mouse kidney. American Journal of Physiology-Renal Physiology. 2007, Jan; 292: F456-F466.

41. Zhou Z, Gong Q, January CT. Correction of defective protein trafficking of a mutant HERG potassium channel in human Long QT Syndrome pharmacological and temperature effects. The Journal of Biological Chemistry. 1999, Oct; 274: 31123-31126.

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Acknowledgements:

I would like to extend a special thank you to the following who have supported me throughout the thesis process.

In particular, I would like to especially thank my thesis advisor, Professor Louise E.O. Darling, throughout my undergraduate research career at Wellesley College. It has always been a pleasure. To work in the lab for the past two and a half years has greatly influenced my passion in the sciences and research.

For the constructive feedback and advice, I would like to thank my thesis committee members: Professor John Goss and Professor Melissa Beers. These experimental ideas would not have been complete without you.

An additional thank you to the Biological Sciences Department and the Biochemistry Department for making this thesis possible.

And lastly, but not least, I would like to thank the Darling Lab members for the wonderful experience: Yeon Joo Lee (’15), Amanda Papakryikos (’14), Medeea Popescu (’17), Myfawny Adams (’16)—[Cameron Lab], Sunny Lew (’15), Kathy Chung (’15), Priya Patel (’16), Heidi Wade (’16), Laurel Kinman (’18) and Stephanie Kim (’18).